The ability to detect binding between phage and mammalian cells is an essential component for discovery of therapeutic and diagnostic antibodies. A typical pipeline for identifying potential therapeutic and diagnostic antibodies includes: (1) phage display and phage panning experiments on soluble protein or cellular associated proteins (in the soluble form or expressed on cells); (2) a phage ELISA performed on soluble protein (for cellular targets—a peptide or protein-mimic of the cellular associated protein); (3) the display gene in the phage genome is subcloned via molecular biology techniques to a soluble antibody fragment expressing plasmid; (4) The antibody fragment then is expressed and purified; (5) once purified the antibody fragment can be tested for cellular functional binding in ELISA, FACS, Guava or FMAT; (6) The lead antibody fragment is analyzed for binding kinetics; and (7) the top antibody lead is then cloned into a full antibody expression vector for large scale production, kinetic analysis and in vivo efficacy models.
Typical assays for analysis of functional binding of phage to protein targets associated with cells include whole mammalian or bacterial cell enzyme-linked immunosorbent assay (ELISA), flow cytometry (Fluorescence Activated Cell Sorter, FACS), Guava microcytometry products (Guava Technologies, Hayward, Calif.), and fluorescence microassay technology (FMAT). ELISAs have high background binding of phage, because cells are complex and phages have a tendency to bind non-specifically. Background binding in ELISA is intensified due to amplification of the binding signal. Cellular ELISAs are also difficult due to the need of many washes between steps, which is cumbersome if the cells are non-adherent as a centrifugal spin is required between each wash. Often adherent cells must be fixed in order to keep the cells attached to the ELISA plate during washes, either manually or on a plate washer. The fixation can change the natural epitopes of the protein on cells. Phage binding in FACs and Guava is also difficult, because each phage clone needs to be purified to get enough phage for a signal.
Currently, most researchers spend a lot of time and effort in cloning the display on the phage to fragments and/or full IgGs in order to investigate the functional binding to cells. The more time spent identifying potential therapeutic antibodies, the longer it takes to get effective therapeutic antibodies into medical clinics. Thus, there is a need in the phage display field for a quick route to identifying functional binding of antibodies to mammalian cells.